A genetic system was developed in Escherichia coil that uses combinatorial genetics to identify mutations in ribosomal RNA (rRNA) drug targets that might lead to antibiotic resistance. Recently, the 16S RNA from Mycobacterium tuberculosis was substituted for E. coil 16S RNA in this system but the construct produced inactive ribosomes when expressed in E. coll. Hybrid 16S rRNAs containing the 5' and central domains from E. coil and the 3' major and minor domains from M. tuberculosis, however, produce active ribosomes in E. coil This suggests that nucleotide differences in the 5' and/or central domain of M. tuberculosis 16S RNA are responsible for loss of function when expressed in E. coll. Absence of function in 30S subunits composed entirely of M. tuberculosis 16S rRNA is probably due to the inability of a nucleotide(s) in M. tuberculosis 16S RNA to interact with an E. coil 30S ligand(s). The goal of this project is to develop genetic technology for the isolation of new anti-infectives that address the issue of drug resistance in M. tuberculosis. Two aims are proposed: (1) The nucleotides in M. tuberculosis rRNA responsible for loss of function in E. coil will be identified and (2) the M. tuberculosis 30S ligand(s) required for expression of M. tuberculosis 16S RNA in E. coil will be identified and cloned. Co-expression of M. tuberculosis 16S RNA and the ligands should produce functional ribosomes containing M. tuberculosis 16S RNA in E. coil Drug resistance in M. tuberculosis is due primarily to chromosomal mutations in the drug targets. Multi-drug resistance appears to occur through sequential accumulation of such mutations. For target-site mutations to be clinically significant, the mutated target must retain most of its biological activity since loss of function decreases the fitness and virulence of the pathogen. This is especially so for functional regions of rRNA, which are critical for protein synthesis. Successful completion of this project will provide a technology to develop novel anti-infectives that recognize all possible functional forms of the target, even if not yet found in nature, and are therefore unlikely to be susceptible to the development of resistance through target modification. Once developed, this technology will allow the use of rRNA genes from other microbial pathogens in designing new anti-infectives.